Ion-exchange chromatography is a method of separating a target analyte by utilizing electrostatic interactions between ions in the target analyte and ion-exchange groups in column packing.
Ion-exchange chromatography is particularly excellent in separation of biomacromolecules such as nucleic acids, proteins, and polysaccharides, and thus has been used in the fields of biochemistry, medicine, and the like.
Ion-exchange chromatography is categorized into anion-exchange chromatography and cation-exchange chromatography. Anion-exchange chromatography can separate anionic substances using cationic column packing. In contrast, cation-exchange chromatography can separate cationic substances using anionic column packing.
Examples of cationic functional groups in anion-exchange column packing include weak cationic groups such as a diethylaminoethyl group and strong cationic groups such as a quaternary ammonium group. Anion-exchange column packing containing these cationic functional groups has been commercially available and used in various research fields.
A nucleic acid is a biomacromolecule comprising chains of nucleotides linked by phosphoester bonds, each nucleotide consisting of a base, a sugar, and a phosphoric acid. There are two types of nucleic acids, i.e., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), depending on the sugar structure.
Ion-exchange chromatographic separation of a target nucleic acid such as a PCR-amplified product, a restriction enzyme fragment of the PCR-amplified product, or a restriction enzyme fragment of a nucleic acid is performed by anion-exchange liquid chromatography that uses phosphate negative charges in the target nucleic acid molecule. This allows separation and detection of each target nucleic acid such as the PCR-amplified product or the nucleic acid fragment by chain length.
Although gel electrophoresis is widely used as a method of separating nucleic acid chains by chain length, it requires complicated operations and long measurement time, thus leaving much room for improvement. Non-Patent Literature 1 discloses a method of separating nucleic acid-related compounds by high-performance liquid chromatography. This method allows separation and detection of nucleic acid chains by chain length in a short time without requiring complicated operations. However, even with this method, it is still difficult to fully separate nucleic acid chains of a similar length. Hence, further improvement in separation performance is required.